Figure 7.

PI31 monomers are sufficient to inhibit 20S proteasome activity. Mass photometry analysis of PI31 dimerization using purified, recombinant (A) hPI31^WT and (B) PI31^V6R dimerization mutant proteins. Proteins were diluted to 40 nM total monomer (1.2 ng μl⁻¹) in PBS during recording and analyzed as described in Experimental Procedures. C, analysis of PI31 (WT, V6R, 152–271) dimerization by chemical cross-linking. Protein, 23 μM, was cross-linked with 0.05% glutaraldehyde or buffer controls as described in Experimental Procedures. One microgram of denatured proteins was separated by SDS-PAGE and blotted against PI31 antibodies. D, inhibition of proteasome activity by hPI31^WT, hPI31^V6R, and hPI31^52–271. 20S proteasome (50 nM) was activated by 0.03% SDS prior to incubation with indicated amounts of total PI31 subunits, [M]_T (where [M]_T = [monomer] = 2 x [dimer]), up to 10-fold molar excess. Proteasome activities were measured in duplicate as described in Experimental Procedures and expressed as percentages relative to controls lacking PI31.